Not Applicable
Not Applicable
The present invention relates to scaleable methods of isolating clinical grade plasmid DNA from microbial cells. The exemplified methods described herein outline a scaleable, economically favorable protocol for the purification of clinical grade plasmid DNA from E. coli which does not rely on expensive chromatography steps during downstream processing of the plasmid preparation, thus making this methodology especially amenable to large scale commercial plasmid purification procedures.
Advances in the areas of gene therapy and DNA vaccination have created a need for the large scale manufacture and purification of clinical-grade plasmid DNA. As pointed out in a recent review (Prazeres, et al., 1999, TIBTech 17: 169-174), despite previous work on small scale plasmid DNA purification methodology, it has been difficult to scale up the manufacture and purification of clinical-grade plasmid DNA. Especially problematic have been downstream processing steps, which for the most part have relied on alkaline lysis of the harvested cells, followed by ammonium acetate precipitation and further downstream processing steps relying heavily on size exclusion, anion exchange and reversed phase chromatography steps. In addition, it should be noted that the expense of raw materials, such as resins and buffers, for multiple chromatographic steps become prohibitive due high unit cost and poor capacity for the large DNA molecules. It is known that the cationic detergent CTAB and various forms of silica have been used for the small scale plasmid DNA preparations and not designed to produce clinical grade plasmid vaccine. The ability of these steps to remove certain impurities has not been recognized nor has their utility for scalable process design. Del Sal et al. (1989, BioTechniques 7(5): 514-519) and Gustincich et al. (1991, BioTechniques 11(3): 298-301) use CTAB to precipitate plasmid DNA from clarified small scale E. coli lysates and genomic DNA from small scale preparations of whole human blood, respectively. Ishaq et al. (1990, Biotechniques 9(1): 19-24) disclose the application of small scale CTAB-precipitated plasmid DNA to a PZ523 spin column, yielding a purified product which is at least suitable as a template for subcloning and dideoxy sequencing. None of this art teaches or suggests the use of detergent-based precipitation steps to produce clinical grade lots of DNA plasmid.
Vogelstein and Gillespie (1979, Proc. Natl. Acad. Sci. USA. 76(2): 615-619) disclose a technique for separating restriction enzyme digests of DNA from agarose gels, in which DNA in the presence of concentrated sodium iodide is bound to glass (silica), washed with ethanol, and eluted at a low salt concentration. Boom et al. (1990, J. Clin. Microbiol. 28(3): 495-503) and Carter and Milton (1993, Nucleic Acids Res. 21(4): 1044) disclose methods for the isolation of plasmid DNA which is suitable for DNA sequencing. Plasmid DNA in the presence of the chaotropic agent guanidinium thiocyanate is bound to silica in the form of diatomaceous earth. The immobilized plasmid DNA is washed with ethanol and eluted at low salt concentrations. Subtle variations of this technique are disclosed in (1) PCT Publication WO 91/10331; (2) PCT Publication WO 98/04730, as well as (3) U.S. Pat. No. 5,075,430, issued to Little on Dec. 24, 1999, which discloses a method of isolating plasmid DNA which depends upon adsorption of the DNA onto diatomaceous earth in the presence of a chaotropic agent followed by separation and elution of the DNA; and (4) U.S. Pat. No. 5,808,041, issued to Padhye et al. on Sep. 15, 1998, which discloses a method of nucleic acid isolation utilizing a composition comprising silica gel and glass particles in the presence of a chaotropic agent. Again, these techniques have not been successfully applied to methodology for large scale DNA plasmid preparations required for generation of gram quantities of plasmid DNA for clinical grade formulations for administration to humans and other potential hosts.
U.S. Pat. No. 4,923,978, issued to McCormick on May 8, 1990, discloses the use of silica to purify DNA by preferentially binding proteinaceous materials.
U.S. Pat. No. 5,576,196, issued to Horn et al. on Nov. 19, 1996, discloses the use of silica to purify DNA by preferentially binding RNA.
U.S Pat. Nos. 5,523,392 and 5,525,319, issued to Woodard et al. on Jun. 4, 1996 and Jun. 11, 1996, respectively, disclose boron silicates, phosphosilicates, and aluminum silicates which can be used as binding surfaces for DNA purification.
PCT International Application PCT/US96/20034 (International publication number WO 98/01464) discloses the use of hydrated calcium silicate to selectively separate organic compounds from biological fluids, such as blood.
Again, none of the above-identified references provide adequate guidance to the artisan of ordinary skill to provide a methodology to prepare scalable, clinical grade DNA plasmid lots which are substantially free of host cell protein, host cell endotoxin, genomic DNA, genomic RNA and plasmid degradates such as linear and open circle forms. To this end, it would be extremely useful to identify a scaleable plasmid purification process which eliminates the requirement of prohibitively expensive chromatography steps while also providing for gram quantities of a DNA plasmid preparation which is clinical grade for use in at least human vaccination and human gene therapy applications. The present invention addresses and meets these needs by disclosing a scaleable plasmid purification process which preferably utilizes a cationic detergent such as CTAB to selectively precipitate plasmid DNA in an upstream step in combination with downstream large scale batch adsorption steps using hydrated, crystalline calcium silicate (herein, xe2x80x9chcCaSiO3xe2x80x9d) or any similar acting compound to remove remaining contaminants such as genomic DNA, genomic RNA, protein, host endotoxin and plasmid degradates such as linear and open circle forms.
The present invention relates to methods of isolating clinical-grade plasmid DNA from microbial cells, methods representing a scaleable, economical manufacturing process which provides alternatives for production and purification of large scale, clinical-grade plasmid DNA. The present invention relates further to several post-lysis core processes which contribute to the scaleable, economical nature of the DNA plasmid purification process. More specifically, post-lysis steps include, but are not limited to, (1) a two part precipitation/dissolution step were plasmid DNA is precipitated with a detergent (such as CTAB) either in a single or stepwise fashion, coupled with concentration and selective dissolution of the CTAB-precipitate plasmid DNA with a salt solution; (2) removal of endotoxin and other remaining impurities by adsorption onto hydrated, crystallized calcium silicate (hcCaSiO3), again, either in a single or stepwise fashion; and; (3) concentration of the purified plasmid DNA by alcoholic precipitation (including but not limited to ethanol, methanol and isopropanol), or another concentrating method, including but not limited to ultrafiltration. These steps may be used in combination, in further combination with additional purification steps known in the art, and/or wherein at least one of the above-mentioned steps is omitted, preferably in combination with other methodology known in the art to be associated with DNA plasmid purification technology.
The methods of the present invention allow for clinical grade DNA plasmid purification from microbial cells including but not limited to bacterial cells, plant cells, yeast, baculovirus, with E. coli being the preferred micorbial host. The clinical grade plasmid DNA purified by the methods described herein is extremely useful for administration to humans as a vaccine or gene therapy vehicle.
An advantage of the plasmid purification process of the present invention is in part due to the finding that stepwise precipitation of DNA with CTAB in conjunction with removal of remaining impurities by adsorption onto hcCaSiO3 removes problematic impurities, including genomic DNA, RNA and DNA degradates such as linear DNA, with a heretofore unrecognized selectivity. A complete process design incorporating these precipitation/purification steps is at the core of the invention disclosed herein. The disclosed process is also scalable.
Another advantage of the purification process of the present invention is the elimination of the need for costly polymer-based chromatography resins through the alternative approach of selective precipitation and adsorption for large scale plasmid preparations.
Another advantage of the purification process of the present invention is that it is fundamentally amenable to manufacturing scale operation. The unit operations consist of precipitation, filtration, adsorption and drying. The use of diatomaceous earth affords an incompressible filter cake while avoiding fouling problems often associated with fermentation products.
Another advantage of the purification process of the present invention is that it avoids the need for adding recombinant RNase, an expensive enzyme, for the removal of RNA at more or more steps during the process.
Another advantage of the purification process of the present invention is that precipitation with a long chain detergent such as CTAB affords reductions in downstream processing volumes which are important in the disposal of solvent containing waste streams at the manufacturing scale.
Yet another advantage of the purification process of the present invention is that alcohol (such as ethanolic) precipitation is an ideal way to gain a stable bulk product which can be resuspended at high concentrations without the anticipated shear damage which occurs during membrane based concentration.
It is an object of the present invention to provide a cost effective process for the large scale purification of clinical grade plasmid DNA from prokaryotic hosts such as E. coli. 
It is further an object of the present invention to provide for post-lysis steps which result in scaleable, economic process for the large scale (i.e., scaleable) purification of plasmid DNA, including but not limited to the post-lysis steps of (i) precipitation of plasmid DNA with a detergent (such as CTAB) either in a single or stepwise fashion, coupled with concentration and selective dissolution of the CTAB-precipitate plasmid DNA with a salt solution; (ii) removal of endotoxin and other remaining impurities by adsorption onto hydrated, crystallized calcium silicate (hcCaSiO3) in either in a single or stepwise fashion; and/or, (iii) concentration of the purified plasmid DNA by alcohol (such as ethanol precipitation) or another concentrating method, including but not limited to ultrafiltration. These steps may be used in combination, in further combination with additional purification steps known in the art, and/or wherein at least one of the above-mentioned steps is omitted, preferably in combination with other methodology known in the art to be associated with DNA plasmid purification technology.
It is a further object of the present invention to provide methods for a cost effective process for large scale (i.e., scaleable) purification of clinical grade plasmid DNA from prokaryotic hosts which comprises the steps of: (i) cell lysis; (ii) lysate clarification with diatomaceous earth-aided filtration; (iii) selective precipitation of plasmid DNA using cetyltrimethylammonium bromide (CTAB), followed by filtration to recover a plasmid DNA-containing filter cake; (iv) selective dissolution of the plasmid DNA-containing filter cake with salt solution; (v) adsorption of residual impurities onto calcium silicate hydrate followed by filtration; and (vi) precipitation of purified plasmid DNA using alcohol (including but not limited to alcohol).
As used interchangeably herein, the terms xe2x80x9cclinical grade plasmid DNAxe2x80x9d and xe2x80x9cpharmaceutical grade plasmid DNAxe2x80x9d refer to a preparation of plasmid DNA isolated from prokaryotic cells which is of a level of purity acceptable for administration to humans for any known prophylactic or therapeutic indication, including but not limited to gene therapy applications and DNA vaccination applications.
As used herein, xe2x80x9cnon-supercoiled plasmid DNAxe2x80x9d refers to any DNA that is not supercoiled plasmid DNA, including any other form of plasmid DNA such as nicked open circle and linear as well as host genomic DNA.
As used herein, xe2x80x9cCTABxe2x80x9d refers toxe2x80x94hexadecyltrimethylammonium bromidexe2x80x94orxe2x80x94cetyltrimethylammonium bromidexe2x80x94.
As used herein, xe2x80x9chcCaSiO3xe2x80x9d refers toxe2x80x94hydrated, crystalline calcium silicatexe2x80x94.
As used herein, xe2x80x9cSTET bufferxe2x80x9d refers to a buffer comprising approximately 50 mM Tris-HCl (xcx9cpH 7.0-9.0), about 50-100 mM EDTA, about 8% Sucrose, and about 2% Triton(copyright)-X100.
As used herein, xe2x80x9cIPAxe2x80x9d refers toxe2x80x94isopropanolxe2x80x94.
As used herein, xe2x80x9cPEGxe2x80x9d refers toxe2x80x94polyethylene glycolxe2x80x94.
As used herein, xe2x80x9cgDNAxe2x80x9d refers toxe2x80x94genomic DNAxe2x80x94.
As used herein, xe2x80x9cgRNAxe2x80x9d refers toxe2x80x94genomic RNAxe2x80x94.
As used herein, xe2x80x9cLRA(trademark)xe2x80x9d refers toxe2x80x94lipid removal agent(trademark).
As used herein, xe2x80x9cEDTAxe2x80x9d refers toxe2x80x94ethylenediaminetetraacetic acidxe2x80x94.
As used herein, xe2x80x9cSCxe2x80x9d refers toxe2x80x94supercoiledxe2x80x94.
As used herein, xe2x80x9cOCxe2x80x9d refers toxe2x80x94open circularxe2x80x94.
As used herein, xe2x80x9cNTUxe2x80x9d refers toxe2x80x94normalized turbidity unitsxe2x80x94.
As used herein, xe2x80x9cLxe2x80x9d refers toxe2x80x94litersxe2x80x94.
As used herein, xe2x80x9cHPLCxe2x80x9d refers toxe2x80x94high performance liquid chromatographyxe2x80x94.